Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada.
PLoS One. 2011;6(11):e27600. doi: 10.1371/journal.pone.0027600. Epub 2011 Nov 16.
Paternal exposures to cancer chemotherapeutics or environmental chemicals may have adverse effects on progeny outcome that are manifested in the preimplantation embryo. The objectives of this study were to determine the impact of paternal exposure to cyclophosphamide, an anticancer alkylating agent, on the formation, chromatin origin and function of micronuclei in cleavage stage rat embryos. Male Sprague-Dawley rats were gavaged with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females to collect pronuclear zygotes and 2 to 8 cell embryos. Micronuclear chromatin structure was characterized using confocal microscopy to detect immunoreactivities for H3K9me3, a marker for maternal chromatin, and lamin B, a nuclear membrane marker. DNA synthesis was monitored using EdU (5-ethynyl-2'-deoxyuridine) incorporation. Fertilization by cyclophosphamide-exposed spermatozoa led to a dramatic elevation in micronuclei in cleavage stage embryos (control embryos: 1% to 5%; embryos sired by treated males: 70%). The formation of micronuclei occurred during the first zygotic division and was associated with a subsequent developmental delay. The absence of H3K9me3 indicated that these micronuclei were of paternal origin. The micronuclei had incomplete peri-nuclear and peri-nucleolar lamin B1 membrane formation but incorporated EdU into DNA to the same extent as the main nucleus. The formation of micronuclei in response to the presence of a damaged paternal genome may play a role in increasing the rate of embryo loss that is associated with the use of assisted reproductive technologies, parenthood among cancer survivors, and paternal aging.
父体暴露于癌症化疗药物或环境化学物质可能会对前胚胎期胚胎的后代产生不利影响。本研究的目的是确定父体暴露于环磷酰胺（一种抗癌烷化剂）对卵裂期大鼠胚胎中微核的形成、染色质起源和功能的影响。雄性 Sprague-Dawley 大鼠连续 4 周灌胃生理盐水或环磷酰胺（6mg/kg/天），并与自然发情的雌性大鼠交配，收集原核受精卵和 2-8 细胞胚胎。使用共聚焦显微镜检测 H3K9me3（母源染色质标志物）和核膜标志物 lamin B 的免疫反应性，以表征微核染色质结构。通过 EdU（5-乙炔基-2'-脱氧尿苷）掺入来监测 DNA 合成。环磷酰胺处理的精子受精导致卵裂期胚胎中微核显著增加（对照组胚胎：1%-5%；处理雄性后代胚胎：70%）。微核的形成发生在第一次卵裂期，随后出现发育延迟。缺乏 H3K9me3 表明这些微核来自父系。微核的核周和核周 lamin B1 膜形成不完全，但与主核一样将 EdU 掺入 DNA。在受损父本基因组存在的情况下形成微核可能在增加与辅助生殖技术、癌症幸存者的生育能力和父本衰老相关的胚胎丢失率方面发挥作用。
